Borrelia spirochetes disseminate from the site of inoculation, an arthropod bite, to multiple tissues, and often persist despite the host immune response, indicating that interactions with and adaptation to the mammalian environment are critical during infection. The Lyme disease spirochete Borrelia burgdorferi (Bb) produces a surface protein, P66, that is a porin and a ligand for several integrins. P66 is required for Bb infection of mice. Dp66 mutant bacteria, however, survive, replicate, and alter protein production at levels equivalent to the parental strain both in vitro and in the protective dialysis membrane chambers in rat peritoneal cavities. The Dp66 strains are not more susceptible than the parental wild-type strain (WT) to any mediator of innate immunity tested to date. Complementation of the mutants by restoration of p66 to the endogenous locus on the chromosome restores infectivity in mice. The integrin-binding activity, specifically, of P66 is not required for Bb infection of mice, but is required for efficient extravasation and dissemination. The absence of P66 or specific amino acid changes in the integrin-binding domain reduces binding affinity to integrin avb3, transendothelial migration of the bacteria in vitro and in vivo, and significantly reduced rates of dissemination in mice. Surface localization on the bacterial cell and the porin function of P66 are not affected by these changes. Thus, although, the absence of integrin binding activity of P66 does affect some virulence attributes of Bb, it does not account for the avirulence of the Dp66 strains. Our hypothesis is that the porin function of P66 is critical to Borrelia survival in the murine host. To test this hypothesis, we will employ c-Myc-tagged p66 mutants that do and do not display deficits in porin function in vitro. All of the c-Myc-P66 proteins are appropriately localized to the bacterial surface, and in this exploratory/developmental project, the recombinant B. burgdorferi strains producing the c-Myc-P66 proteins will be characterized in vivo with regard to survival and dissemination in mice. We predict that porin-deficient mutants will be attenuated or avirulent in mice. We are in the exclusive position of having the experience, strains, and infection models to determine whether the porin function of the integral outer membrane protein P66 of Borrelia plays a significant role during murine infection, and to progress to a clearer mechanistic understanding of how the multifunctional protein P66 enables the bacteria to establish infection and disseminate in mammalian hosts. This work will advance our understanding of the pathogenic mechanisms of Borrelia spirochetes, and may guide investigations into the environmental challenges faced by Borrelia at the site of inoculation and perhaps future development of novel therapeutics (e.g. pore blockers) specifically against Borrelia infections.